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Received: 16 July 2018 Accepted: 9 October 2018 Published: xx xx xxxx
White-nose syndrome is associated with increased replication of a naturally persisting coronaviruses in bats Christina M. Davy1,2, Michael E. Donaldson1, Sonu Subudhi 3, Noreen Rapin3, Lisa Warnecke4,5, James M. Turner4,6, Trent K. Bollinger7, Christopher J. Kyle8, Nicole A. S.-Y. Dorville4, Emma L. Kunkel4, Kaleigh J. O. Norquay4, Yvonne A. Dzal4, Craig K. R. Willis4 & Vikram Misra 3 Spillover of viruses from bats to other animals may be associated with increased contact between them, as well as increased shedding of viruses by bats. Here, we tested the prediction that little brown bats (Myotis lucifugus) co-infected with the M. lucifugus coronavirus (Myl-CoV) and with Pseudogymnoascus destructans (Pd), the fungus that causes bat white-nose syndrome (WNS), exhibit different disease severity, viral shedding and molecular responses than bats infected with only Myl-CoV or only P. destructans. We took advantage of the natural persistence of Myl-CoV in bats that were experimentally inoculated with P. destructans in a previous study. Here, we show that the intestines of virus-infected bats that were also infected with fungus contained on average 60-fold more viral RNA than bats with virus alone. Increased viral RNA in the intestines correlated with the severity of fungus-related pathology. Additionally, the intestines of bats infected with fungus exhibited different expression of mitogen-activated protein kinase pathway and cytokine related transcripts, irrespective of viral presence. Levels of coronavirus antibodies were also higher in fungal-infected bats. Our results suggest that the systemic effects of WNS may down-regulate anti-viral responses in bats persistently infected with M. lucifugus coronavirus and increase the potential of virus shedding. Bats are hosts for many viruses and are thought to be the source of some viruses that have spilled over to humans and other mammals, causing fatal disease. These include coronaviruses causing severe acute respiratory syndrome (SARS1), Middle East respiratory syndrome (MERS2–5), porcine epidemic diarrhoea (PED6) and swine acute diarrhoea syndrome (SADS7); paramyxoviruses such as Hendra8 and Nipah9; and filoviruses like Marburg10 and Ebola11. Four families of viruses that are pathogenic for other mammalian species (Coronaviridae12, Paramyxoviridae13, Rhabdoviridae14 and Filoviridae15) may also have originated in bats. These viruses often cause serious disease in their secondary hosts, but most do not appear to cause clinical signs or pathology in bats16–18, suggesting that uniquely benign relationships have co-evolved between the viruses and their primary bat hosts19,20. While relatively little is known about the dynamics of viral infections in bats, these viruses may be maintained in bat populations as a result of either persistently infected individuals, reinfection after waning immunity, or spatial transmission dynamics21,22. 1
Environmental and Life Sciences Graduate Program, Trent University, Peterborough, ON, Canada. 2Ontario Ministry of Natural Resources and Forestry, Wildlife Research and Monitoring Section, Trent University, Peterborough, ON, Canada. 3Department of Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. 4Department of Biology, University of Winnipeg, Manitoba, Canada. 5Present address: Department of Animal Ecology and Conservation, University Hamburg, Hamburg, Hamburg, Germany. 6 Present address: Institute for Land Water and Society, Charles Sturt University, Albury, New South Wales, Australia. 7 Canadian Wildlife Health Cooperative and Department of Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. 8Forensic Science Department, Trent University, Peterborough, ON, Canada. Christina M. Davy, Michael E. Donaldson, Sonu Subudhi and Noreen Rapin contributed equally. Correspondence and requests for materials should be addressed to V.M. (email:
[email protected]) SCIENTIFIC ReporTs | (2018) 8:15508 | DOI:10.1038/s41598-018-33975-x
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www.nature.com/scientificreports/ The rare spill-over of bat viruses to other animals may require a “perfect storm” of conditions that include increased contact between bats or fomites and other mammals, possibly due to human impacts on habitat quality23, and the ability of the virus to infect, replicate, and transmit in the secondary host. The rate of viral shedding and the amount of detectable virus associated with bat colonies fluctuates, with periodic increases often linked to parturition, waning maternal immunity, nutritional stress or increased energy consumption17,24–29. Increased shedding of virus by a colony of bats may reflect an increase in the proportion and number of susceptible individuals, or an increase in the replication of persistent or latent virus normally suppressed by the host. For herpesviruses, reactivation from latency is linked to perturbations caused by a variety of physiological, immunological and psychological stressors30. The mechanisms that trigger the reactivation of latent or persistently infecting viruses are not clearly understood, but the increased shedding of viruses is correlated with some incidents of spill-over of bat viruses to other animals31. The Canadian prairies are home to three species of bats, including the little brown bat (Myotis lucifugus), big brown bat (Eptesicus fuscus), and northern long-eared bat (Myotis septentrionalis). All three species hibernate from October to May, sometimes in shared hibernacula. We recently demonstrated that ~30% of hibernating M. lucifugus sampled over two years from hibernacula in Manitoba were infected with a coronavirus (Myl-CoV), which persisted at low levels in the intestine32. A closely related coronavirus also infects E. fuscus33. Whereas bats appear to be relatively resistant to viral infections, a cold-adapted fungus that was recently introduced to North America has caused widespread mortality in some species of bats in eastern United States and Canada34–37. The fungus (Pseudogymnoascus destructans) causes white-nose syndrome (WNS) in hibernating bats, which is characterized by the growth of white fungal mycelia on the face and exposed skin of the wings and tail membranes. The visual and microscopic effects of P. destructans on the skin of the wings are associated with increased expression of several genes devoted to innate immunity and inflammation in wing tissue38,39. Profound systemic effects include dehydration, hypovolemia, metabolic acidosis, and fat depletion, which can lead to death40–42. Other systemic effects of bat WNS include an accumulation of neutrophils in the lungs, which is accompanied by an increase in the expression of several cytokine genes43 suggesting that even the most severely afflicted hibernating bats are capable of at least some systemic immune response to fungal infection. Previous studies on other species have demonstrated that a fungus and a virus could interact during co-infection and affect each other44,45. Similar interactive impacts of co-infection with P. destructans and viruses on bat immune responses are not known. We used M. lucifugus experimentally-infected with P. destructans and/ or naturally infected with Myl-CoV as a model to understand how co-infections influence bat-virus interactions. This system allows us to avoid confounding factors of direct pathogen-pathogen interactions, because the fungus affects the skin, while the coronavirus infections occur internally, almost exclusively in the ileum and lungs32. We hypothesized that co-infection would alter the molecular response of bats to a persistent viral infection, and that viral shedding would change as a result of the increased or disrupted host immune response. To test this prediction, we examined tissues collected from M. lucifugus at the termination of an earlier study that quantified the effects and pathogenesis of P. destructans in hibernating bats experimentally infected with the fungus37, some of which were naturally infected with Myl-CoV32. This combination of uninfected, virus-infected, fungus-infected and co-infected M. lucifugus allowed us to test our hypothesis that host responses to co-infection are synergistic and not simply additive.
Results
Quantitation of Myl-CoV and M. lucifugus RNA through reverse transcription quantitative PCR (RT-qPCR) and dual-RNA-sequencing indicated that co-infected bats had significantly higher levels of Myl-CoV RNA than bats infected with virus alone. The amount of Myl-CoV RNA correlated with the severity of WNS pathology in co-infected bats. This phenomenon was associated with specific molecular responses to co-infection, even in the intestines of bats where only one of the two pathogens was directly interacting with the host tissue. The levels of antibodies against Myl-CoV nucleocapsid (N) protein were also higher in co-infected bats. Each key result is discussed in detail below.
Bats co-infected with the fungus P. destructans and the virus Myl-CoV contained higher levels of Myl-CoV RNA. Myl-CoV genomic RNA was detected in bats infected only with Myl-CoV (virus-infected;
7/18), co-infected bats (European P. destructans (3/13), or with North American P. destructans (7/16)37). There was no difference in the frequency of Myl-CoV detected among these treatments (p-value = 0.801). We pooled bats infected with the two P. destructans isolates for all further analyses and tested whether co-infection with P. destructans and Myl-CoV correlated with an increase in viral replication. Our RT-qPCR data showed that the co-infected bats contained 60-fold more Myl-CoV RNA on average than the virus-infected bats (Mann Whitney test; p-value = 0.014; Fig. 1). Relative quantities of Myl-CoV RNA detected in the ileum of the virus-infected bats were low and showed low variation (Standard Deviation of 1/ΔCT = 0.005), compared to the relative quantities of Myl-CoV RNA in the co-infected bats (Standard Deviation of 1/ΔCT = 0.108; Fig. 1). The severity of WNS fungal pathology varied in co-infected bats, and we therefore tested whether relative quantities of viral RNA in the ileum correlated with the severity of WNS symptoms. Levels of WNS severity were scored based on fungal hyphae on the wings, secondary bacteria in wing lesions, oedema, necrosis and inflammation in wing lesions, and levels of neutrophils in lung, spleen and liver interstitium. Severity scores for wing tissue, secondary bacteria in lesions, and neutrophils in the lung interstitium positively correlated with relative amounts of coronavirus RNA in hibernating bats (Table 1).
Bat responses to co-infection exceed the sum of responses to virus or fungal infection alone. To determine the extent to which Myl-CoV and P. destructans infection interact to influence gene expression in bat intestines, we performed a transcriptomic analysis on bat intestines comparing gene expression among the
SCIENTIFIC ReporTs | (2018) 8:15508 | DOI:10.1038/s41598-018-33975-x
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1/ CT(Myl-CoV-GAPDH)
Level of Coronavirus RNA *p2 fold change, FDR 2 fold change, FDR > 0.05), light grey (2 and false discovery rate (FDR)-corrected p-values
